RESUMO
Autoantibodies specific against fibrillarin, a 34-kD nucleolar protein associated with U3-snRNP, are present in patients with systemic sclerosis (SSc). To understand the mechanisms involved in the induction of these autoantibodies, we prepared a series of human fibrillarin recombinant proteins covering the entire molecule and analyzed their interaction with the autoantibodies present in various connective tissue diseases. Our results showed that antifibrillarin autoantibodies are present not only in SSc, as previously reported, but also in a variety of other connective tissue diseases. Patients with SSc (58%), mixed connective tissue diseases (60%), CREST syndrome (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, and telangiectasia syndrome) (58%), systemic lupus erythematosus (39%), rheumatoid arthritis (60%), and Sjogern's syndrome (84%) showed presence of antifibrillarin autoantibodies. Results obtained from competitive inhibition radioimmunoassay and Western blot analyses with purified recombinant fusion proteins revealed that these autoantibodies react primarily with epitope(s) present in the NH2- (AA 1-80) and COOH-terminal (AA 276-321) domains of fibrillarin. Autoantibodies reacting with internal regions of fibrillarin are less frequent. Analysis of the hydrophilicity profiles of reactive peptides showed presence of three potential antigenic sites in the NH2- and two in the COOH-terminal regions. While a hexapeptide sequence NH2 terminus of fibrillarin is shared with an Epstein-Barr virus-encoded nuclear antigen, the COOH-terminal region shares sequence homology with P40, the capsid protein encoded by herpes virus type 1. Interestingly, these two regions of fibrillarin also contain the most immunodominant sequences, as predicted by surface probability and the Jameson and Wolf antigenic index. These observations suggest that molecular mimicry might play an important role in the induction of antifibrillarin autoantibodies.
Assuntos
Autoanticorpos/análise , Proteínas Cromossômicas não Histona/imunologia , Doenças do Tecido Conjuntivo/imunologia , Epitopos , Escleroderma Sistêmico/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Sequência de Bases , Proteínas Cromossômicas não Histona/biossíntese , Imunofluorescência , Humanos , Dados de Sequência Molecular , Radioimunoensaio , Proteínas Recombinantes/biossínteseRESUMO
We have generated for the first time monoclonal antibodies (mAbs) specific for topoisomerase I (topo I) from scleroderma patients, and tight skin mice which develop a scleroderma-like syndrome. The epitope specificity of these antibodies has been determined using a series of fusion proteins containing contiguous portions of topo I polypeptide. Western blot analysis demonstrated that both human and mouse mAbs bound strongly to fusion protein C encompassing the NH2-terminal portion of the enzyme, and weakly to fusion proteins F and G containing regions close to the COOH-terminal end of the molecule. This crossreactivity is related to a tripeptide sequence homology in F, G, and C fusion proteins. It is interesting that a pentapeptide sequence homologous to that in fusion protein C was identified in the UL70 protein of cytomegalovirus, suggesting that activation of autoreactive B cell clones by molecular mimicry is possible. Both human and mouse mAbs exhibiting the same antigen specificity, also share an interspecies cross-reactive idiotope. These data suggest that B cell clones producing antitopo autoantibodies present in human and mouse repertoire are conserved during phylogeny, and are activated during the development of scleroderma disease.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , DNA Topoisomerases Tipo I/imunologia , Escleroderma Sistêmico/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Western Blotting , DNA Topoisomerases Tipo I/química , Epitopos , Camundongos , Camundongos Mutantes/imunologia , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Autoantibodies against nuclear proteins like RNA polymerase I (RNA pol I) are produced in a number of rheumatic autoimmune diseases. Production of antibodies specific for the 190-kD subunit of RNA pol I appears to be characteristic in the patients with systemic sclerosis. Previous investigations have shown that the tight skin (TSK) mouse is an experimental model for systemic sclerosis. In the present study we show that the TSK mice produce high titers of anti-RNA pol I antibodies, both of IgM and IgG classes. To characterize the immunochemical properties of these antibodies we obtained a large panel of hybridomas from these mice. Analysis of these hybridomas revealed that clonal frequency of autoreactive B cells specific for RNA pol I are higher in the TSK mice that in the controls. mAbs obtained from the TSK mice were specific for the 190-kD subunit and cross-reacted with Escherichia coli and phage T7 RNA polymerases (155-, 150-, and 107-kD polypeptides). We have also demonstrated that these antibodies bind better to the phosphorylated enzymes. The anti-RNA pol I mAbs were divided into three groups in terms of their functional property. The first group of antibodies increased the catalytic activity of the enzyme whereas the antibodies of the second group inhibited the enzymatic activity. Competitive inhibition RIAs showed that these two groups of antibodies bound to distinct epitopes. The third group of antibodies was neutral and had no activity on the enzyme function. These results suggest that TSK mouse anti-RNA pol I antibodies recognize three or more conserved epitopes. To understand the molecular basis of the generation of such autoreactive antibodies we analyzed their V gene repertoire. Northern analysis of RNAs of 14 TSK hybridomas showed that the VH genes encoding these antibodies were mainly from VH J558 family. It is possible that these genes were derived from a single germline gene or from a set of related genes of a single subgroup.
Assuntos
Autoanticorpos/sangue , Autoanticorpos/genética , Genes de Imunoglobulinas , Camundongos Mutantes/imunologia , RNA Polimerase I/imunologia , Pele/imunologia , Animais , Anticorpos Monoclonais , Bacteriófago T7/enzimologia , Ligação Competitiva , Western Blotting , Reações Cruzadas , Escherichia coli/enzimologia , Imunofluorescência , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cinética , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica , Fosforilação , RNA Polimerase I/metabolismo , Radioimunoensaio , Esclerose/enzimologia , Esclerose/genética , Esclerose/imunologiaRESUMO
In the previous studies we have shown that tight-skin (TSK) mouse is an experimental model for systemic sclerosis. This mutant mouse develops autoantibodies specific for scleroderma target antigens. To determine whether the expansion of autoantibody repertoire in TSK mouse occurs by selective expansion of certain variable region gene families, and whether CD5+ B cells contribute significantly to the production of autoantibodies, we have analyzed a panel of 60 hybridomas producing autoantibodies specific for scleroderma target autoantigens. Northern analysis of RNAs from these hybridomas showed that 70% were expressing genes from VHJ558 family while genes from 36-09 and J606 families were not at all represented. In contrast, in the cDNA libraries derived from splenic B cells, the expression of VHJ558 and 36-09 gene families were at an expected frequency corresponding to their genomic complexity (44% and 11.6%, respectively). These results demonstrate that there is a strong bias toward the use of J558 genes in TSK mouse autoantibody repertoire. On the other hand the expression of VK gene families was mostly random and corresponded to their frequency in splenic C kappa cDNA library. The pairing of VH:VK genes was stochastic. Analysis of the expression of J segments, however, revealed that JH2 and JK2 were predominantly used in the autoantibodies. Analysis of the expression CD5 mRNA in these hybridomas indicate that CD5+ B cells do not contribute significantly to the autoimmunity in TSK mice. These findings suggest that the expansion of peripheral autoreactive B cells in TSK mouse is determined by their immunoglobulin variable region rather than the genetic properties linked to a particular B cell subset.
Assuntos
Autoanticorpos/genética , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Dermatopatias/imunologia , Animais , Especificidade de Anticorpos , Antígenos CD/análise , Linfócitos B/imunologia , Antígenos CD5 , Camundongos , Dermatopatias/genéticaRESUMO
An enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase is described for the detection and quantitation of anti-influenza virus antibodies. Compared with complement fixation and hemagglutination inhibition tests, ELISA is far superior with respect to sensitivity and reliability. Non-specific viral inhibitors present in sera do not affect the titer in ELISA. Its sensitivity, close to that of radioimmunoassay permits detection of small amounts of antibodies in pulmonary secretions and supernatants from in vitro spleen cell cultures.
Assuntos
Anticorpos Antivirais , Infecções por Orthomyxoviridae/imunologia , Animais , Testes de Fixação de Complemento , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Testes de Inibição da Hemaglutinação , Pulmão/imunologia , Masculino , Coelhos , Baço/imunologiaRESUMO
Tight-skin (TSK) mouse represents an experimental for systemic sclerosis, displaying cutaneous hyperplasia, connective tissue alterations in the internal organs and developing autoantibodies against several scleroderma target autoantigens. TSK mouse syndrome is associated with a mutation in fibrillin-1 (Fbn-1), the major component of 10 nm microfibrils. Here, we have investigated whether TSK mouse develops autoimmunity to Fbn-1 similar to scleroderma target autoantigens. Our results show that anti-Fbn-1 IgG autoantibodies are present in high titer in many TSK mice. Specificity of these antibodies was confirmed by competitive inhibition assays and Western blotting analysis using recombinant human Fbn-1 protein. TSK mouse autoantibodies recognize a conserved epitope present in the C region of Fbn-1. These results indicate the presence of Fbn-1 specific T and B cells in TSK mouse repertoire.
Assuntos
Autoanticorpos/sangue , Doenças Autoimunes/imunologia , Modelos Animais de Doenças , Proteínas dos Microfilamentos/imunologia , Escleroderma Sistêmico/imunologia , Animais , Autoantígenos/imunologia , Western Blotting , Fibrilina-1 , Fibrilinas , Humanos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Radioimunoensaio , Proteínas Recombinantes , Dermatopatias/imunologiaRESUMO
Tight skin (TSK) mice develop cutaneous hyperplasia accompanied by histopathological alterations of skin and collagen metabolism similar to those described in human scleroderma. Diffuse scleroderma, the most severe form of progressive systemic sclerosis, is associated with the production of autoantibodies specific for Scleroderma 70 antigen (topoisomerase I). Our studies show that there is an increase in the level of serum anti-topoisomerase I (topo I) autoantibodies in aged TSK mice. The monoclonal antibodies isolated from TSK mice bind to epitopes which interact with autoantibodies from scleroderma patients. A significant number of TSK monoclonal anti-topo I antibodies and serum immunoglobulin (Ig) from aged TSK mice bear a cross reactive idiotype (Id) recognized by a syngeneic monoclonal anti-Id antibody obtained from a 2 month-old TSK mouse. Analysis of V gene usage by monoclonal anti-topo I antibodies showed that the majority of these antibodies are encoded by VH genes derived from VHJ558 family pairing with VK genes from various families in a stochastic manner.
Assuntos
Autoanticorpos/imunologia , Autoimunidade , DNA Topoisomerases Tipo I/imunologia , Camundongos Endogâmicos/imunologia , Animais , Autoanticorpos/genética , Sítios de Ligação de Anticorpos , Ligação Competitiva , Northern Blotting , Southern Blotting , Células Clonais , Reações Cruzadas , DNA/análise , Sondas de DNA , Expressão Gênica , Genes de Imunoglobulinas , Hibridomas/imunologia , Idiótipos de Imunoglobulinas , Técnicas In Vitro , Camundongos , RNA/análise , Radioimunoensaio , Escleroderma Sistêmico/imunologiaAssuntos
Autoanticorpos/imunologia , DNA Topoisomerases Tipo I/imunologia , Escleroderma Sistêmico/imunologia , Animais , Linfócitos B/imunologia , Modelos Animais de Doenças , Epitopos/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Mutantes , RNA Polimerase I/imunologiaRESUMO
Tight-skin mouse, a mutant strain with a single gene defect, develops cutaneous hyperplasia and specific autoantibodies, like humans affected by scleroderma. The autoantibodies produced in the tight-skin mouse are encoded primarily by heavy-chain variable (VH) genes from the J558 family. To understand the genetic basis of production of autoantibodies, we have analyzed the structure of J558 genes encoding these autoantibodies. The results showed that J558 genes encoding these antibodies were not derived from a selected germ-line gene(s) or a single subfamily but were derived from genes belonging to diverse J558 subfamilies. However, two prototype VH genes representing two new subfamilies were found to be repeatedly expressed in their germ-line form in eight independent clones. Autoantibodies with distinct specificities appear to be generated by pairing of similar/identical VH genes with different V kappa genes derived from the same or different families. Fourteen of 18 autoantibodies shared a conserved heptapeptide sequence motif, YNEKFKG, in the second complementarity-determining region of heavy chains. Usage of germ-line genes from diverse J558 subfamilies bearing a common motif to encode autoantibodies suggests a regulatory role for this motif. Thus, selection and expansion of the autoreactive B-cell repertoire in the tight-skin mouse appear to be VH-gene mediated. The frequency of N nucleotide addition at diversity-joining (D-JH) junctions was lower, whereas the frequency of usage of the DFL16 segment was higher. Finally, in contrast to normal and other autoimmune mouse strains, the frequencies of D-D fusions and D inversions were higher in tight-skin mouse total immunoglobulin as well as autoantibody repertoires.
Assuntos
Autoanticorpos/genética , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Cruzamentos Genéticos , Primers do DNA , Feminino , Variação Genética , Humanos , Masculino , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/imunologia , Homologia de Sequência do Ácido NucleicoRESUMO
Autoantibodies against thymocytes and RBC may contribute to the pathophysiology of homozygous viable motheaten (mev) autoimmune disease. Whether the production of these autoantibodies in mev mouse results from polyclonal nonspecific B cell activation or specific Ag-driven stimulation is not known. To understand the mechanisms involved in the induction of antithymocyte autoantibody response in mev mouse, we have studied the fine antigenic specificity, structure, and origin of three antithymocyte autoantibodies derived from mev splenic B cell hybridomas. Western blot analysis showed that these mAb bind to polypeptides of 33 and 105 kDa present in RBC and thymocytes, respectively. Additional specificities for the epitopes present in other polypeptides distinguished these three autoantibodies. Northern hybridization and flow microfluorimetry analysis indicated that these hybridomas are derived from the Ly1+ B cell subset. These autoreactive Ly-1 B cell hybridomas, chosen on the basis of their specificity, expressed L chain V genes from a single VK family (VK9) and VH genes from J606 and S107 families. Hybridomas UN34.11 and UN42.5 expressed the VK9 gene identical to that used by peritoneal Ly1+ B cells from various mouse strains and malignant B lymphoma cells secreting anti-mouse RBC treated with proteolytic enzyme bromelin and anti-SRBC antibodies. The third hybridoma, S2-14.2, used a VK9 gene identical to that expressed by MOPC41. None of the VK genes encoding these autoantibodies showed any somatic mutations. In the case of VH genes, the two hybridomas UN42.5 and S2-14.2 derived from two separate fusions, used identical VH genes from the J606 family. The third hybridoma UN34.11 used unmutated V11 germline VH gene, a member of the S107 family. Southern hybridizations, using oligonucleotide probes specific for CDR1 and CDR2, showed that the VH genes encoding the J606 autoantibodies were derived from a germline gene found in the 6.7-kb fragment of EcoRI-digested germline DNA. This germline VH gene is distinct from VH22.1 germline gene that codes for antigalactan antibodies. Sequence analysis of this gene showed perfect homology with the rearranged VH genes confirming the lack of somatic mutations. Thus, our data demonstrate that antithymocyte antibody response occurring in mev mouse is polyclonal and it involves Ly-1 B cells expressing unmutated germline VH and VK genes. These results indicate that antigen driven stimulation may not play an important role in the induction of anti-thymocyte antibody response in mev mouse.
Assuntos
Autoanticorpos/genética , Eritrócitos/imunologia , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Camundongos Mutantes/genética , Timo/imunologia , Animais , Antígenos Ly/análise , Linfócitos B/fisiologia , Sequência de Bases , Northern Blotting , Southern Blotting , Cadeias Pesadas de Imunoglobulinas/genética , Ativação Linfocitária , Camundongos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da PolimeraseRESUMO
Cutaneous hyperplasia observed in tight skin mice is due to a mutation located on chromosome 2. While homozygous mice die in utero, the heterozygotes survive. TSK syndrome is associated with the presence of autoantibodies specific for scleroderma target autoantigens. The presence of autoantibodies specific for topoisomerase I is characteristic of both human and murine disease. We have generated two distinct genotypes of mice, TSK/+ and +/+ with respect to the TSK trait by breeding TSK mice with immunodeficient mouse strains. Since the mutated gene of TSK syndrome has not yet been cloned, only histological and biochemical criteria were used for defining TSK genotype. In the F1 mice derived by mating TSK/+ mice with RAG2-/-, JH-/-, or C57BLvit/vit mice, we have found a good correlation between the amount of serum anti-topoisomerase I autoantibodies present and the histopathological and biochemical alterations that are characteristic of TSK scleroderma-like syndrome.
Assuntos
Autoanticorpos/sangue , DNA Topoisomerases Tipo I/imunologia , Escleroderma Sistêmico/imunologia , Pele/patologia , Animais , Colágeno/análise , Hidroxiprolina/análise , Camundongos , Camundongos Endogâmicos C57BL , Escleroderma Sistêmico/patologia , Pele/químicaRESUMO
The tight skin mouse strain has been proposed for use as an animal model of systemic sclerosis because this animal exhibits a condition that has biochemical and pathologic similarities to the human disease. To date, however, evidence of inflammatory and immunologic changes in the tight skin mouse has been scarce. We demonstrated the presence of antinuclear antibodies in approximately half of these mice ages 8 months and older. This suggests that there is an autoimmune component in their disease process. The antibodies were identified as anti-topoisomerase I by a characteristic staining pattern on HEp-2 cells and by Western blot analysis. Except for a low incidence of anti-DNA antibodies, none of the other parameters tested, including mitogen responses, lymphokine production, and anti-erythrocyte antibodies, was indicative of immune system dysregulation.
Assuntos
Autoimunidade , Camundongos Mutantes , Esclerodermia Localizada/imunologia , Animais , Formação de Anticorpos , Autoanticorpos/análise , Western Blotting , Divisão Celular , DNA Topoisomerases Tipo I/análise , Modelos Animais de Doenças , Interleucina-2/metabolismo , Linfócitos/patologia , Camundongos , Esclerodermia Localizada/genética , Esclerodermia Localizada/patologia , PeleRESUMO
The immunoglobulin light chain V kappa 1 gene family is polymorphic in murine inbred strains and this family has been subdivided into five sub-groups (V kappa 1A-E). The V kappa 1A sub-group contributes to approximately 2% of the total serum immunoglobulin light chains in several mouse strains. However, it has been reported that this sub-group is absent in New Zealand Black (NZB) mouse serum. Amino acid sequencing of myeloma proteins from this inbred mouse has shown that they belong to the V kappa 1B sub-group. We report here the structure of nine functional germline genes from NZB mice that have high homologies to the V kappa 1A, V kappa 1B, V kappa 1C, and V kappa 1D sub-groups. In addition, a novel germline gene representing the prototype of a new sub-group (designated V kappa 1F) has been identified. We have isolated different V kappa 1 germline genes from a single restriction fragment length polymorphism (RFLP) fragment, as well as identical V genes from two different RFLP migrating bands. Therefore, the complexity of the genes encoding the immunoglobulin variable region cannot be determined solely by RFLP analysis. Nucleotide sequence analysis of 16 V kappa 1 genes which code for NZB autoantibodies indicate that they belong to five different V kappa 1 sub-groups with five hybridomas (31%) expressing the V kappa 1A sub-group. Comparison of the sequences of V kappa 1 genes expressed in hybridomas with corresponding germline genes show no somatic mutations.
Assuntos
Genes de Imunoglobulinas , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Animais , Sequência de Bases , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos NZB , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Collagen V plays a major regulatory role in the formation of heterotypic fibers of the dermis and cartilaginous tissues as well as in the assembly of extracellular matrix. The pN/pN mouse, which is defective in collagen V alpha 2 gene, exhibits skeletal abnormalities, skin fragility, and alterations in the collagen fiber organization, whereas the TSK/+ mouse, which is defective in fibrillin-1, the major component of microfibrils present in the extracellular matrix, develops cutaneous hyperplasia and autoimmunity. We have studied the role of collagen V in the formation of heterotypic collagen fibers in F1 mice, which are obtained by breeding pN/pN with TSK/+ mice. Our results show that F1 progeny neither develop cutaneous hyperplasia nor produce anti-topoisomerase I autoantibodies, unlike TSK/+ mice. The diameter of the collagen fibrils in the skin is also comparable to that found in control mice. Thus, the phenotypic changes observed in the TSK mouse could be reversed by genetic complementation with a collagen V-defective mouse.
Assuntos
Alelos , Colágeno/genética , Mutagênese Sítio-Dirigida/genética , Pele/patologia , Animais , Autoanticorpos/sangue , Colágeno/análise , DNA Topoisomerases Tipo I/imunologia , Hiperplasia/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fenótipo , Pele/química , Pele/ultraestruturaRESUMO
Tight-skin (TSK) mouse, the experimental model for scleroderma, develops cutaneous hyperplasia, cardiac hypertrophy, pulmonary emphysema and autoimmunity against scleroderma target autoantigens. The cutaneous hyperplasia is associated with the accumulation of microfibrils and elastic fibers in the middle and deep dermis. Fibrillin-1 (Fbn-1) is a major component of the 10-12 nm microfibrils found in the extracellular matrix. In this study we report the identification of a genetic marker in the Fbn-1 gene that can distinguish the mutant phenotype. TSK mice exhibit an unique polymorphism in the Fbn-1 gene. RNA analysis, PCR analysis and sequence determination of the mutant gene showed that the Fbn-1 gene polymorphism is due to intragenic duplication of a segment of the gene coding for 3.0 Kb of mRNA sequence (10 Kb of the genome). Histological analysis of skin samples from F1 progeny obtained by crossing TSK mice with JH-/-, RAG2-/- or vit/vit showed a significant correlation between the inheritance of the defective Fbn-1 gene and the development of cutaneous hyperplasia. Further, our results also show that in mice deficient in mature B cells inheriting the defective Fbn-1 gene, development of cutaneous hyperplasia is not abrogated. Thus, production of autoantibodies or the presence of mature B lymphocytes do not play an integral role in the pathogenesis of cutaneous hyperplasia.
Assuntos
Linfócitos B/fisiologia , Proteínas dos Microfilamentos/genética , Pele/patologia , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Sequência de Bases , DNA/genética , Sondas de DNA , Fibrilina-1 , Fibrilinas , Marcadores Genéticos , Genótipo , Hiperplasia/genética , Hiperplasia/imunologia , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/metabolismo , Pele/imunologia , Dermatopatias/genética , Dermatopatias/imunologiaRESUMO
The tight skin (TSK/+) mouse has been proposed as an experimental model for progressive systemic sclerosis because of the biochemical alterations in collagen synthesis and pathological similarities to the human disease. Here, we report the analysis of tight skin mice sera for the presence of anti-cytoplasmic and anti-nuclear autoantibodies and determination of the frequency of hybridomas producing anti-cellular autoantibodies. The binding specificity of TSK mAbs to nuclear and cytoplasmic antigens such as keratin, actin, vimentin, and mitochondria was determined. Of 71 monoclonal antibodies that we have studied, only 3 appear to bind to foreign as well as self-antigens, indicating that the majority of these antibodies do not belong to the class of natural autoantibodies. Our results also showed that the frequency of hybridomas producing anti-nuclear and anti-cytoplasmic antibodies was higher in TSK mice than in C57BL/6 pa/pa, the control mouse strain, used in these studies. The results of the analysis of V gene usage showed that the majority of anti-cytoplasmic and anti-nuclear antibodies are encoded by genes from a restricted number of VH and VK genes families. In the sera of TSK mice we have detected the presence autoantibodies specific for cytoplasmic antigens in addition to anti-nuclear and anti-topoisomerase I antibodies which are characteristic of scleroderma. Since the presence of anti-cytoplasmic antibodies has not been described in scleroderma, the significance of their production in tight skin mice is not clear. However, the presence of such autoantibodies in the animal model provides a basis for investigation of this type of antibodies in human disease.